70-90% of all eukaryotic proteins are N-acetylated. There are seven established N-acetylated prokaryotic proteins. ESAT-6 (Early secreted antigen, 6kDa) is the only example of an N-acetylated bacterial virulence factor. ESAT-6 is secreted by ESX-1 (ESAT-6 system 1), a key virulence determinant in both Gram-positive and mycobacterial pathogens. The role of N-acetylation (N-Ac) of bacterial proteins in pathogenesis has not been investigated. The ESX-1 substrates require each other for export (coupled secretion). The mechanisms underlying coupled secretion are not known. The result is a fundamental gap in understanding how bacteria cause disease. The long-term goal is to understand the molecular mechanisms that underlie mycobacterial pathogenesis. The overall objective is to identify how N-Ac promotes mycobacterial pathogenesis. The central hypothesis is that N-Ac of ESAT-6 contributes to pathogenesis by directly mediating protein export by the ESX-1 system. The hypothesis is based on the applicant's preliminary data which show that altering N-Ac of ESAT-6 uncouples ESX-1 export and leads to attenuation. Therefore, the following specific aims have been proposed. 1) Establish how N-Ac promotes ESX-1 export 2) Determine how a novel gene in M. marinum pro- motes ESAT-6 acetylation 3) Identify the ESX-1-associated N-acetyltransferase in M. tuberculosis (M. tb). Under Aim 1, the link between N-Ac of ESAT-6 and coupled secretion will be investigated using molecular, biochemical and proteomic approaches. The scope of N-Ac in Mycobacterium will be addressed by identifying all of the N-acetylated proteins in M. marinum and M. tb using proteomics. Under Aim 2, the mechanism of N-Ac of ESAT-6 will be studied in vitro using biochemistry. A complementary genetic approach will identify residues in the putative ESAT-6 N-acetyltransferase required for N-Ac of ESAT-6 and for interaction with the ESX-1 apparatus. Finally, under the Aim 3, the tools generated in the M. marinum system will be used to identify the gene in human M. tb required for N-Ac of ESAT-6. The role of N-Ac in ESX-1 secretion and virulence in M. tb will be investigated. The proposed work will provide the first example of how N-Ac promotes bacterial pathogenesis and will result in an improved understanding of the molecular mechanisms of ESX secretion. This proposal is innovative because coupled secretion, one of the biggest outstanding questions in the ESX-1 field, is being approached by focusing on N-Ac, a likely overlooked protein modification in bacteria. The significance of the research proposed here is the identification and characterization of the first reported mycobacterial mutant strain with uncoupled ESX-1 substrate secretion, which will result in a vertical step in the ESX-1 field. Moreover, this proposal represents the primary step in understanding how N-Ac promotes bacterial protein secretion and pathogenesis, which will likely initiate a new field of study.